Nucleic acids are typically detected by contacting them with labelled probe molecules under controlled conditions and detecting the labels to determine whether specific binding or hybridisation has taken place. Various methods of labeling probes are known in the art, including the use of radioactive atoms, fluorescent dyes, luminescent reagents, electron capture reagents and light absorbing dyes. Each of these labeling systems has features which make it suitable for certain applications and not others. For reasons of safety, interest in non-radioactive labeling systems lead to the widespread commercial development of fluorescent labeling schemes particularly for genetic analysis. Fluorescent labeling schemes permit the labeling of a relatively small number of molecules simultaneously, typically 4 labels can be used simultaneously and possibly up to eight. However the costs of the detection apparatus and the difficulties of analysing the resultant signals limit the number of labels that can be used simultaneously in a fluorescence detection scheme.
More recently there has been development in the area of mass spectrometry as a method of detecting labels that are cleavably attached to their associated probe molecules. Until recently, Mass Spectrometry has been used to detect analyte ions or their fragment ions directly, however for many applications such as nucleic acid analysis, the structure of the analyte can be determined from indirect labeling. This is advantageous particularly with respect to the use of mass spectrometry because complex biomolecules such as DNA have complex mass spectra and are detected with relatively poor sensitivity. Indirect detection means that an associated label molecule can be used to identify the original analyte, where the label is designed for sensitive detection and a simple mass spectrum. Simple mass spectra mean that multiple labels can be used to analyse multiple analytes simultaneously. In fact, many more labels than can currently be used simultaneously in fluorescence based assays can be generated.
WO98/31830 describes arrays of nucleic acid probes covalently attached to cleavable labels that are detectable by mass spectrometry which identify the sequence of the covalently linked nucleic acid probe. The labeled probes of this application have the structure Nu-L-M where Nu is a nucleic acid covalently linked to L, a cleavable linker, covalently linked to M, a mass label. Preferred cleavable linkers in this application cleave within the ion source of the mass spectrometer. Preferred mass labels are substituted poly-aryl ethers. These application discloses a variety of ionisation methods and analysis by quadrupole mass analysers, TOF analysers and magnetic sector instruments as specific methods of analysing mass labels by mass spectrometry.
WO 95/04160 disclose ligands, and specifically nucleic acids, cleavably linked to mass tag molecules. Preferred cleavable linkers are photo-cleavable. These application discloses Matrix Assisted Laser Desorption Ionisation (MALDI) Time of Flight (TOF) mass spectrometry as a specific method of analysing mass labels by mass spectrometry.
WO 98/26095 discloses releasable non-volatile mass-label molecules. In preferred embodiments these labels comprise polymers, particularly biopolymers, and more particularly nucleic acids, which are cleavably attached to a reactive group or ligand, i.e. a probe. Preferred cleavable linkers appear to be chemically or enzymatically cleavable. This application discloses MALDI TOF mass spectrometry as a specific method of analysing mass labels by mass spectrometry.
WO 97/27327, WO 97/27325, WO 97/27331 disclose ligands, and specifically nucleic acids, cleavably linked to mass tag molecules. Preferred cleavable linkers appear to be chemically or photo-cleavable. These application discloses a variety of ionisation methods and analysis by quadrupole mass analysers, TOF analysers and magnetic sector instruments as specific methods of analysing mass labels by mass spectrometry.
WO 01/68664 and WO 03/025576 disclose organic molecule mass markers that are analysed by tandem mass spectrometry. These applications disclose mass markers comprised of two components, a mass tag component and a mass normalization component that are connected to each other by a collision cleavable group. Sets of tags can be synthesised where the sum of the masses of the two components produce markers with the same overall mass. The mass markers are typically analysed after cleavage from their analyte. Analysis takes place in an instrument capable of tandem mass spectrometric analysis. In the first stage of analysis, the MS/MS instrument is set to select ions with the mass-to-charge ratio that corresponds to the mass marker comprising both the mass tag and mass normaliser, which may be referred to as the ‘parent ion’. This selection process effected by the MS/MS instrument allows the markers to be abstracted from the background. Collision of selected the marker ions in the second stage of the analysis separates the two components of the tag from each other. Only the mass tag fragments of the parent ion, which may be referred to as the ‘daughter ions’ are detected in the third stage of analysis. This allows confirmation that the ion selected in the first stage of analysis is from a mass marker and not from a contaminating ion, which happens to have the same mass-to-charge ratio as the parent ion. The whole process greatly enhances the signal to noise ratio of the analysis and improves sensitivity. This mass marker design also compresses the mass range over which an array of mass markers is spread as mass markers can have the same mass as long as they give rise to mass tag fragments that are uniquely resolvable. Moreover, with isotopes, this mass marker design allows the synthesis of markers, which are chemically identical, have the same mass but which are still resolvable by mass spectrometry. Use of these markers to identify oligonucleotide probes is described.
Thus, the prior art provides oligonucleotide probes cleavably linked to tags that are detectable by mass spectrometry. The prior art also shows that these probes enable multiplexing of nucleic acid probe binding assays. However, multiplexed assays require more than just multiple tags. Many nucleic acid probe binding assays do not function well when multiplexed because of problems of cross-hybridisation. This is a particular problem for polymerase chain reaction (PCR) based assays, for which it is very costly and time-consuming to optimize reactions involving multiple primer pairs. The problems are due to the high risk of cross hybridization of primers to incorrect templates leading to cross-amplification of templates and hence to incorrect results.
However, some nucleic acid probe binding assay methods that enable high-order multiplexing are known in the art. Most notably, Oligonucleotide Ligation Assays (OLA) such as those described in U.S. Pat. No. 4,988,617, which discloses an assay for determining the sequence of a region of a target nucleic acid, which has a known possible mutation in at least one nucleotide position in the sequence. In this sort of assay, two oligonucleotide probes that are complementary to immediately adjacent segments of a target DNA or RNA molecule which, contains the possible mutation(s) near the segment joint, are hybridised to the target DNA. A ligase is then added to the juxtaposed hybridised probes. Assay conditions are selected such that when the target nucleotide is correctly base paired, the probes will be covalently joined by the ligase, and if not correctly base paired due to a mismatching nucleotide(s) near the segment joint, the probes are incapable of being covalently joined by the ligase. The presence or absence of ligation is detected as an indication of the sequence of the target nucleotide.
Similar assays are disclosed in EP-A-185 494. In this method, however, the formation of a ligation product depends on the capability of two adjacent probes to hybridize under high stringency conditions rather than on the requirement of correct base-pairing in the joint region for the ligase to function properly as in the above U.S. Pat. No. 4,988,617. Other references relating to ligase-assisted detection are, e.g., EP-A-330 308, EP-A-324 616, EP-A-473 155, EP-A-336 731, U.S. Pat. No. 4,883,750 and U.S. Pat. No. 5,242,794.
Ligation mediated assays have a number of advantages over conventional hybridization based assays. The reaction is more specific than hybridization as it requires several independent events to take place to give rise to a signal. Ligation reactions rely on the spatial juxtaposition of two separate probe sequences on a target sequence, and this is unlikely to occur in the absence of the appropriate target molecule even under non-stringent reaction conditions. This means that standardised reaction conditions can be used enabling automation. In addition, due to the substrate requirements of ligases, incorrectly hybridised probes with terminal mismatches at the ligation junction are ligated with very poor efficiency. This means that allelic sequence variants can be distinguished with suitably designed probes. The ligation event creates a unique molecule, not previously present in the assay which enables a variety of useful signal generation systems to be employed to detect the event. This high specificity makes ligation based assays easier to multiplex as disclosed in provisional U.S. application 20030108913.
Further improvements in stringency and multiplexing can be achieved using circularising probes. Circularising probes comprise a single oligonucleotide probe, typically about 70 nucleotides in length or greater, in which the two probe sequences that are to be ligated to each other are located at either end of the probe molecule. The probe sequences are designed so that when they bind to their target sequence, the two probe sequences are brought into juxtaposition. The probe sequences can then be ligated to form a closed circular loop of DNA. Since both probe sequences are linked to each other, when one probe sequence binds to its target, binding of the second probe sequence takes place with rapid kinetics. This ensures that intra-molecular ligation is much more likely than inter-molecular ligation reducing cross-ligation of probes to very low levels. In addition, cross-ligated probes are still linear and it is highly unlikely that two or more probes will cross-ligate to form a circular species. Similarly, mismatched probes, i.e. probes that have bound to a target that does not exactly match the probe sequence, are unable to ligate and therefore will not be circularized. This all means that correctly reacted probes can be distinguished from incorrectly reacted probes by the fact that correctly reacted probes are circular. The ability to resolve correctly matched probes means that large numbers of probes can be used simultaneously in a single reaction. The key to using circularizing probes lies in being able to obtain a signal from circularised probes rather than from non-circularised probes and various methods have been disclosed in the prior art to date.
The first disclosure of circularizing probes appears to have been made by Aono Toshiya in JP 4262799 and JP 430-4900. These applications both disclose the use of ligation reactions with circularising probes.
Circularisation is detected by the ability of circularized probes to undergo linear Rolling Circle Amplification (RCA). The methodology disclosed in the above Japanese applications comprises contacting the sample in the presence of a ligase with a probe oligonucleotide. Correctly hybridised probes will be circularized by ligation and will act as a template in a RCA polymerization reaction. A primer, which is at least partially complementary to the circularised probe, together with a strand-displacing nucleic acid polymerase and nucleotide triphosphates are added to the circularized sequences and a single stranded nucleic acid is formed which has a tandemly repeated sequence complementary to the circularized probe and at least partially to the template. The amplification product is then detected either via a labelled nucleotide triphosphate incorporated in the amplification, or by an added labelled nucleic acid probe capable of hybridizing to the amplification product.
Other methods based on RCA of circularized probes have been disclosed in U.S. Pat. No. 5,854,033 and related divisions of this application published as U.S. Pat. No. 6,344,329, U.S. Pat. No. 6,210,884 and U.S. Pat. No. 6,183,960. The most notable difference between the disclosure of these applications and the disclosure of JP 4262799 and JP 430-4900, is the use of hyper-branching RCA. In this method, a second primer that is at least partially complementary to the single-stranded product of linear RCA of a circularized probe is added to the reaction. This results in a further geometric amplification of the single stranded product.
Another method for resolving circularized probes from non-circularised probes is disclosed in WO 95/22623. The methods disclosed in this application exploit the fact that circularized probes are not susceptible to degradation by exonucleases while unreacted linear probes are susceptible to degradation. In addition, cyclisation of a probe ‘locks’, the probe onto its target, i.e. the probes are resistant to being separated from their target. This allows circularized probes to be distinguished from linear probes by subjecting the probes to non-hybridising conditions. This approach to the use of circularizing probes is sometimes referred to as Padlock Probe technology.
Despite the ability of mass tags to enable multiplexing of nucleic acid assays, none of the prior art on mass tags provides methods of analysing nucleic acids using circularising probes. Similarly, none of the prior art on circularising probes provides methods of detecting circularising probes suggests using mass spectrometry. It is thus an object of this invention to provide methods and reagents to exploit the abilities of both mass tags and circularising probes to be used in highly multiplexed nucleic acid detection assays.